单分子荧光原位杂交(smFISH)技术及应用
作者:
作者单位:

1)昆明理工大学省部共建非人灵长类生物医学国家重点实验室,昆明 650500;2)深圳湾实验室生物影像实验室,深圳 518107;3)昆明理工大学生命科学与技术学院,昆明 650500

作者简介:

孙正龙 Tel: 13584804523, E-mail: sunzl@lpbr.cn关淼 Tel: 13584851257, E-mail: mguan2021@hotmail.comSUN Zheng-Long. Tel: 86-13584804523, E-mail: sunzl@lpbr.cnGUAN Miao. Tel: 86-13584851257, E-mail: mguan2021@hotmail.com

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基金项目:

云南省自然科学基金(202001BC070001,202102AA100053), 深圳湾实验室开放基金(SZBL2021080601012) 和深圳湾实验室- 仪景通公司光学显微成像技术开发项目资助。


Technique and Application of Single-molecule Fluorescence in situ Hybridization
Author:
Affiliation:

1)State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming 650500, China;2)Bio-imaging Lab, Shenzhen Bay Laboratory, Shenzhen 518107, China;3)Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China

Fund Project:

This work was supported by grants from The Natural Science Foundation of Yunnan Province (202001BC070001, 202102AA100053), Shenzhen Bay Laboratory Open Program (SZBL2021080601012), and Shenzhen Bay Laboratory-Evident Company Cooperation Fund.

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    摘要:

    单分子荧光原位杂交(single-molecule fluorescence in situ hybridization,smFISH)技术是一种通过用偶联荧光基团的寡核苷酸探针,对固定细胞或组织中单个mRNA分子进行成像的方法。smFISH可对RNA进行定位、定量,以此对目标转录本进行实时研究。smFISH适用于细胞、组织切片等多种类型生物样本。近年来,多种基于基础smFISH的改进技术被发明,进一步促进了该技术的实际应用。smFISH良好的RNA单分子可视化能力,使得其在发育生物学、神经生物学及肿瘤生物学等基础生物学科中得到了广泛的应用。本文综述了smFISH技术基本原理、smFISH技术的局限性、smFISH衍生技术方法、smFISH在不同生物学科中的应用进展,并对smFISH技术的发展前景做出展望。

    Abstract:

    Single molecule fluorescence in situ hybridization (smFISH) is a method for imaging single mRNA molecule in fixed cell or tissue using oligonucleotide probes coupled with fluorophores. It can realize real-time study of interested transcripts by RNA localization and quantification. smFISH is widely suitable for many types of biological samples such as cell and tissue sections. It was invented in 1982 which opened up the application of visualizing single molecules. However, due to its shortcomings such as poor binding specificity, Raj et al. optimized this technique in 2008, using 48 independent probes that were separately coupled with fluorophores to locate transcripts. In contrast, methods using multiple labeled probes can distinguish false positive or false negative results due to a single probe misbinding or unbinding event. However, with the continuous application of the technique, it was found that the scheme still has many technical defects, such as low probe specificity, weak fluorescence intensity, low hybridization efficiency, and high background fluorescence. Since then, a series of derivative technologies have been developed. For example, HCR-FISH is a multi-fluorescence in situ hybridization method based on orthogonal amplification and hybridization chain reaction, which significantly improves the problem of weak signal. SeqFISH amplifies the signal and reduces nonspecific binding by continuously hybridizing the mRNA in the cell, imaging it, and stripping the probe in order to barcode RNA. MERFISH utilizes combination labeling, continuous imaging and other technologies to increase detection throughput, and uses binary barcodes to offset single-molecule labeling and detection errors, with more advanced built-in error correction functions to effectively improve the accuracy of results. ClampFISH uses biological orthogonal click chemistry to effectively lock the probe around the target and prevent the probe from disengaging in amplification microscopy. RNAscope amplifies its own signal while simultaneously suppressing the background by using novel probe design strategy and hybridization-based signal amplification system. Split-FISH uses splitting probes for signal enhancement to accurately detect single RNA molecule in complex tissue environments. AmpFISH achieves imaging of short RNA molecules by preparing long single-strand DNA concatemers through controlled rolling circle amplification. CircFISH uses two unique sets of probes (PC probes and PL probes) to distinguish between linear and circular RNAs. π-FISH rainbow enables simultaneous detection of DNA, RNA, and proteins at the single-molecule level with π-FISH target probes. HT-smFISH is more suitable for large or high throughput form of systematic experiments. With the development of technology, the subsequent data analysis process is particularly important. Different analysis software, such as dotdotdot and FISH-quant v2, also improve the process of smFISH. The excellent ability of smFISH to visualize single molecule of RNA makes that it is widely used in basic biological disciplines such as tumor biology, developmental biology, neurobiology, botany, virology. In this paper, we reviewed the basic principle of smFISH technology, its development process and improvement, limitations of smFISH technology and how to avoid them, its derivative technologies include HCR-FISH, SeqFISH, MERFISH, ClampFISH, RNAscope, Split-FISH, AmpFISH, CircFISH, π-FISH rainbow and HT-smFISH. The application progress of smFISH in different biological disciplines, such as developmental biology, tumor biology, neurobiology. Finally, the development prospect of smFISH technology is prospected.

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芮涵,孙正龙,关淼.单分子荧光原位杂交(smFISH)技术及应用[J].生物化学与生物物理进展,2024,51(6):1239-1255

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历史
  • 收稿日期:2023-07-12
  • 最后修改日期:2024-05-29
  • 接受日期:2023-11-15
  • 在线发布日期: 2024-06-25
  • 出版日期: 2024-06-20